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標題: | AtERF1及AIL7在非生物性逆境耐受性上分別扮演的角色 Roles of AtERF1 and AIL7 in Abiotic Stress Tolerance |
作者: | Wei-Wen Kuo 郭瑋文 |
指導教授: | 林讚標 |
關鍵字: | 阿拉伯芥,非生物性逆境,耐受性, Arabidopsis,Abiotic stress,Tolerance, |
出版年 : | 2010 |
學位: | 碩士 |
摘要: | 環境中的許多不利因子皆會影響植物的生長,其中非生物性逆境諸如乾旱、高鹽、高低溫等。以乾旱逆境為例,在水分缺乏的情形下會干擾植物的正常生長,然而植物會利用各種生理與生化機制來抵抗乾旱而達到生存的目的。前人研究顯示植物會於轉錄層面進行基因的調控,促使下游基因使之活化或抑制以因應環境逆境。我們在2008年9月從Dr. Wolfgang Droge-Laser 實驗室獲得ERF轉錄因子的大量表現之阿拉伯芥種子。而在前人的研究已知一些ERF轉錄因子會參予在非生物性逆境的調控上,然而還有許多成員都還不清楚其功能。所以我們篩選具有抗旱的植株並收其後代種子而進行這些轉錄因子之功能研究。經由定序結果比對後得到2個阿拉伯芥的基因,Ethylene Response Factor 1 (ERF1)及 Aintegumenta-like 7 (AIL7)。此轉錄因子皆屬於AP2/ERF superfamily的成員,分別含有一個及二個高度保留性的AP2 DNA binding domain。根據eFP的資料庫數據得知,ERF1會受高鹽所誘導,尤其在根部的位置;而AIL7則會受乾旱、高鹽及滲透壓所誘導,但在高低溫逆境下則無誘導表現。此外,ERF1及AIL7皆不受ABA誘導表現,故可能參予在ABA-independent pathway。首先,我們測試了在高鹽底下的種子發芽率,發現二個大量表現轉殖品系發芽率都較野生型品系高出許多;接著觀察在高鹽環境下的根系發展情形,結果顯示出野生型阿拉伯芥的根長都較二個突變株來的短;而進一步直接使用鹽水澆灌觀察其存活率,有趣的是我們看到了在ERF1的大量表現轉殖株有耐鹽的情形;然而在AIL7片段基因大量表現的轉殖株卻有相反的結果,也就是對鹽逆境較為敏感。最後我們處理缺水逆境也看到了ERF1轉殖株有較高的存活率;反之,AIL7轉殖株的存活植株相較野生型植株來的低許多。而我們構築了ERF1及AIL7與GFP的結合蛋白,透過原生質體表現後,在CaMV35S啟動子調控下,可看到綠色螢光訊號位於細胞核中。在loss-of-function研究中,T-DNA insertion knockout突變株與野生型植株間並沒有看到有明顯的差異性。因為篩選到的AIL7大量表現轉植株只有插入部分的片段,所以我們利用了轉錄活性分析探討其功能。短暫表現轉錄活性分析結果顯示全長(1/1497)及C端片段(688/1497)的AIL7並無轉錄活性;而N端部分片段(175/687)卻有顯著的轉錄活性。根據以上實驗結果我們首度發現在非生物性逆境下ERF1扮演著正向調控角色;而部分片段的AIL7是具有轉錄上的活性,並且在非生物性逆境下扮演著負調控的角色。 Various abiotic stresses including drought, high salinity, and extreme temperatures affect plant growth. To survive, plants have to react and adapt to these stresses. Exposure to these unfavorable conditions leads to various physiological and biochemical alternations in the process of acquiring stress tolerance. Regulation of genes at transcriptional level has been described in response to environmental stresses. Particularly, many transcription factor (TF) genes are stress inducible, and function in regulating stress signal transduction pathways. Here we screened ERF TF overexpressed transgenic seeds by drought stress, and two lines, containing Ethylene Response Factor 1 (ERF1) and Aintegumenta-Like 7 (AIL7), showed transgene respectively phenotype from the WT plants. These two genes have highly conserved AP2 DNA binding domain and are induced by salt and/or osmotic stresses but not by abscisic acid (ABA), implicated they are involved in the ABA-independent pathway. We have found that the germination rate and root elongation in these transgenics are greater than those of wild type. In salt and drought stress experiments, ERF1 overexpression (OE) transgenic plants were more tolerant to salt and drought stresses, but AIL7 partial fragment OE transgenic plants were more sensitive. Localization assay of ERF1-GFP and AIL7-GFP showed ERF1 and AIL7 were located in the nucleus. Loss-of-function study of T-DNA knockout mutants did not show significant differences from wild-type plant. Functional domain study of AIL7 by the transient expression assay exhibited that full length (1/1497) and C-terminal (688/1497) of AIL7 had no transcriptional activity, while N-terminal partial fragment (175/687) showed distinct transcriptional activity. These results suggested that ERF1 may play a positive role to enhance tolerance in abiotic stress, while partial fragment of AIL7 have transcriptional activity and act as a repressor in abiotic stress. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/47228 |
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顯示於系所單位: | 植物科學研究所 |
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